An effective assay reacts to positive samples with a range of responses distinct and separate from the responses resulting from negative samples. Variation in response measurements causes greater spread in the distributions of responses to both positive and negative samples, increasing the likelihood that these distributions will overlap. Increased overlap in the response to positive and negative signals degrades assay performance resulting in reduced sensitivity and specificity.
In multiplexed arrays such as the multiplexed PCR liquid array assay, there are many possible sources of variation. Variability in the efficiency of the nucleic acid extraction, the extent of PCR amplification, and hybridization of amplified product to the probe-microsphere conjugates affect the median fluorescent intensity (MFI) measurements [see reference 15]. Non-specific interactions between the primers and variability in the binding of the fluorophore to the probes results can result in disparity in the responses to negative samples. The composition of the sample matrix (e.g., blood, serum, saliva etc) may also affect the measurement responses for both positive and negative samples as well. In addition to these effects, which can be seen in measurements made by a single laboratory or operator, there are sources of variation owing to differences between laboratories, such as differences in thermal cycler ramp times, ambient temperatures, and the manual techniques used by various operators.
Therefore, a problem with current techniques is that responses generated during a multiplexed assay such as a PCR diagnostic assay using liquid array detection are subject to multiple sources of variation. Variation in the median fluorescent intensity (MFI) responses results in elevated threshold values and thus reduced true positive rates.